System and method for rapid analysis of polymer additives

ABSTRACT

The subject technology is directed to a CO 2 -based chromatography system and method for rapid determination of the levels and/or the presence or absence of polymer additives (PAs) leachable or extractable from packing materials or implantable medical devices.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/044,853, filed Oct. 2, 2013, which claims the priority benefit ofU.S. Provisional Application No. 61/744,889, filed Oct. 3, 2012, theentire content of which is incorporated herein by reference in itsentirety as though fully set forth herein.

FIELD

The subject technology relates to CO₂-based chromatography; inparticular, the subject technology relates to a CO₂-based chromatographysystem (“CO₂-based system”) and method for analysis of polymer additivesleachable and/or extractable from packing materials or implantablemedical devices.

BACKGROUND

The polymer additives (PAs) which may migrate from the packagingmaterials are of concern to manufacturers and suppliers of containersfor the heavily regulated pharmaceutical, food and medical deviceindustries. Due to these regulations packaging material manufacturersare directed to control and monitor their products to ensure that noharmful additives are used, which could migrate from these products andcause injuries to the health of the consumers.

Certain rules promulgated by the U.S. Food and Drug Administrationmandate the need for adequate information related to packagingmaterials. An FDA rule, currently in force, states a drug is deemed tobe adulterated “if its container is composed, in whole or in part, ofany poisonous or deleterious substance which may render the contentsinjurious to health . . . .” Under this rule, in certain instances, theFDA may require a quantitative extraction profile be obtained for thepackaging materials used so as to identify and quantify each detectablesubstance that may migrate from the packaging materials to their food ordrug contents.

The current methods for obtaining a qualitative or quantitativeextraction profile include HPLC (high performance or pressure liquidchromatography) and GC (gas chromatography). However, there areshortcomings associated with each of these methods. For example, the GCmethods only detect volatile compounds and non-volatile compoundsrequire derivatization prior to a GC analysis, which is burdensome,expensive and time-consuming. In LC methods, although no samplederivatization is required, the typical run time of a sample on an HPLCinstrument is about 25 minutes; which has recently been reduced to about10 minutes by using a UHPLC (ultrahigh performance or pressurechromatography) instrument. However, there are several disadvantages tousing HPLC and UHPLC, one of which is their using of toxic organicsolvents as mobile phase and generating toxic waste, which is expensiveto purchase and dispose of.

The use of non-toxic Supercritical CO₂ (SC-CO₂) as an alternative toorganic solvents as the mobile phase has resulted in the advent ofsupercritical fluid chromatography (SFC) which embraces many of thefeatures of liquid and gas chromatography. Theoretically, SC-CO₂provides a low viscosity mobile phase that achieves higher diffusionrates and enhanced mass transfer over the solvents used in HPLC.However, the current SFC instruments (which are mainly retooled HPLCs)and methods have many limitations including, for example, long samplerun time, inaccurate or imprecise control over the mobile phase densityand composition, inability to reliably deliver modifiers at low amounts(<5% of liquid CO₂), susceptibility to system pressure fluctuations andsample backflow, baseline noise, sample carryover, and lack ofrobustness, which prevent users from rapidly obtaining reproducibleresults.

Therefore, there still remains a need for a more improved chromatographysystem and method that can overcome the above limitations and allow fora rapid and robust analysis of the polymer additives extractable and/orleachable from packaging materials or implantable medical devices.

SUMMARY

The subject technology is, in part, based on a discovery that many PAscan be conveniently and reproducibly analyzed in a short period of time(e.g., in less than about 4 minutes) by a C02-based chromatographymethod and system involving, inter alia, a stationary phase withparticle sizes of about 0.5 to 3.5 μm in diameter, a CO₂ mobile phasewith a pre-column dwell volume of about 75 μl to about 500 μl. Themethod and system of the subject technology generate high resolutionchromatograms which allow for accurate detection and quantification ofPAs.

In addition, the subject technology is based, in part, on the surprisingdiscovery that the CO₂-based system of the subject technology iscompatible with all extraction solvents used in the present disclosure,eliminating the need for solvent evaporation and reconstitution prior toanalysis. This compatibility of the CO₂-based system of the subjecttechnology with various sample solutions or extraction solvents savesuser's time and reduces sample preparation efforts.

The subject technology is illustrated, for example, according to variousaspects described below.

In one aspect, the subject technology relates to a method for detectingone or more polymer additives (PAs) in a sample by means of a CO2-basedchromatography analysis including: (1) providing a sample including oneor more PAs for analysis; wherein the sample is prepared with, extractedor dissolved in a diluent including at least 60% organic solvent, withthe proviso that the sample is analyzed without a solvent exchange stepor is not subject to a solvent exchange step once it is prepared orbefore analysis by the method of the subject technology; (2) applyingthe sample to a chromatography column with a solid stationary phaseincluding inorganic or hybrid particles having a mean particle size ofabout 0.5 to about 3.5 microns, wherein said particles have a polar,non-polar or polar/non-polar surface functionality, and wherein theparticles retain said one or more PAs; (3) eluting the one or more PAsfrom the chromatography column by a mobile phase including a mixture ofliquid CO2 and a modifier to form one or more eluted PAs, wherein themobile phase has dwell volume of about 75 μL to about 500 μL; and (4)detecting said one or more eluted PAs by a suitable detector or adetection device.

In an embodiment relating to this or any other aspects of the subjecttechnology, the sample is not subject to a derivatization step. Inanother related embodiment, the particles having a non-polar surfacefunctionality include capped particles with non-polar surface modifiersincluding an alkyl group, alkenyl group, alkynyl group, aryl group, analkyl or aryl group containing one or more embedded non-polarfunctionalities, or a mixture thereof. In another related embodiment,the particles having a polar/non-polar surface functionality includeuncapped particles with free surface hydroxyl groups and non-polarsurface modifiers including an alkyl group, alkenyl group, alkynylgroup, aryl group, an alkyl or aryl group containing one or moreembedded non-polar functionalities, or a mixture thereof. In anotherrelated embodiment, the particles having a polar surface functionalityinclude capped or uncapped particles with polar surface modifierscomprising hydroxyl, aldehyde, amine, ester, ketone, halogen, boron,phosphorus or sulphur, carbonyl, imine, oxime, N-oxide, diol, carboxy,nitrile, azide, diazonium, isonitrile, cyanate, isocyanate, or analoguesof the aforementioned O-containing groups. In another relatedembodiment, the particles have a mean particle size of about 0.5 toabout 2 microns. In another related embodiment, the particles have amean pore volume in the range of about 0.1 to about 2.5 cm/g. In anotherrelated embodiment, the particles have a mean pore diameter in the rangeof about 100 to about 1000 Angstroms. In another related embodiment, theinorganic particles include silicon, aluminum, titanium, cerium,zirconium, barium, cobalt, copper, europium, gadolinium, iron, nickel,samarium, silver, titanium, diamond, zinc, boron or oxides thereof,silicon carbide, carbon black, carbon nanotubes, ceramic, glass,metallic materials or mixtures thereof. In another related embodiment,the hybrid particles include an inorganic portion and an organicportion. In another related embodiment, the inorganic portion of thehybrid particles includes silicon, aluminum, titanium, cerium,zirconium, barium, cobalt, copper, europium, gadolinium, iron, nickel,samarium, silver, titanium, diamond, zinc, boron or oxides thereof,silicon carbide, carbon black, carbon nanotubes, ceramic, glass,metallic materials or mixtures thereof. In another related embodiment,the organic portion of the hybrid particles includes substituted orunsubstituted C1-C18 alkane, alkylene, alkenylene, alkynylene or arylenemoiety bonded to one or more atoms of the inorganic portion. In anotherrelated embodiment, the organic portion of the hybrid particles includessubstituted or unsubstituted C1-C18 alkylene, alkenylene, alkynylene orarylene moiety bridging two or more atoms of the inorganic portion. Inanother related embodiment, the chromatography column is kept in atemperature range of about 5° C. to about 85° C. In another relatedembodiment, the modifier added to the liquid CO2 in a constant orgradient mode or both over an elution period or a fraction thereof. Inanother related embodiment, the modifier is a polar water-miscibleorganic solvent including methanol, ethanol or isopropanol,acetonitrile, acetone, tetrahydrofuran, mixtures thereof, and mixturesof water and any of these solvents. In another related embodiment, thegradient mode includes increasing or decreasing flow volume of themodifier. In another related embodiment, the elution period is less than5 minutes. In another related embodiment, the gradient mode includesincreasing the flow volume of the modifier from about 0% to about 50%(v/v CO2) or any intervals therebetween. In another related embodiment,the gradient mode includes increasing the flow volume of the modifierfrom about 0% to about 25% (v/v CO2). In another related embodiment, theliquid CO2 is in a supercritical state or subcritical state or both. Inanother related embodiment, the detection includes determining thelevels or the presence or absence of the one or more PAs. In anotherrelated embodiment, the detection is by way of a mass spectrometer;Evaporative Light Scattering (ELS) detector, Circular Dichroism (CD)detector, Flame Ionization Detector (FID) or a photodiode array detector(PDA). In another related embodiment, the sample includes a packagingmaterial or a solution or an extract thereof. In another relatedembodiment, the chromatography column is part of a chromatography systemcomprising a pre-column mobile phase dwell volume of about 320 μL;wherein said pre-column mobile phase dwell volume is the volume of themobile phase present in a fluidic connection between a junction at whichthe CO2 and the modifier are mixed and the head of the chromatographycolumn. In another related embodiment, the one or more PAs are elutedfrom the chromatography column by the mobile phase with a flow rate ofabout 1 to 4 mL/min. In another related embodiment, the chromatographycolumn has a length of about 50 to 150 mm and an internal diameter about2 to 4 mm. In another related embodiment, the sample is not subject to aderivatization step because the method of the subject technology issufficiently sensitive to detect minute amounts of PAs in the sample.

In another aspect, the subject technology relates to a chromatographymethod for detecting one or more PAs including: (1) providing a samplecontaining one or more PAs for analysis; wherein the sample is preparedwith, extracted or dissolved in a diluent including at least 60% organicsolvent, with the proviso that the sample is not subject to a solventexchange step; (2) applying the sample to a chromatography systemincluding: (a) a column with a solid stationary phase comprising aninorganic or hybrid particle having a mean particle size of about 0.5 toabout 3.5 microns, wherein said particle has a polar, non-polar orpolar/non-polar surface functionality, wherein said column has a lengthof about 50 to 150 mm and an internal diameter about 2 to 4 mm, andwherein the solid stationary phase retains said one or more PAs; (b) apre-column mobile phase dwell volume of about 75 μL to about 500 μL;wherein said pre-column dwell volume includes a volume or space within afluidic connection between a junction at which two or more mobile phasesolvents including CO2 and a modifier are mixed to the head of thecolumn; and (c) a post-column mobile phase dwell volume of about 10 μLto about 450 μL; wherein said pose-column dwell volume includes a volumeor space within a tubular connection between the end of the column and adetector; (3) eluting the one or more PAs from the chromatography columnby a mobile phase including a mixture of CO2 and a modifier to form oneor more eluted PAs, wherein the mobile phase has a flow rate of about 1to 4 mL/min; and (4) detecting said one or more eluted PAs.

In another aspect, the subject technology relates a kit for performinganalysis or detecting one or more PAs in a sample including: (1) asample preparation device for preparing the sample containing one ormore PAs for analysis; wherein the sample is prepared with, extracted ordissolved in a diluent comprising at least about 60% organic solvent,with the proviso that the sample is analyzed without a solvent exchangestep or is not subject to a solvent exchange step; (2) a chromatographycolumn with a solid stationary phase including inorganic or hybridparticles having a mean particle size of 0.5 to 3.5 microns; whereinsaid particles have a polar, non-polar or polar/non-polar surfacefunctionality and retain said one or more PAs; and (3) one or morestandards for calibrating and facilitating the analysis and detection ofthe one or more PAs.

Additional features and advantages of the subject technology will be setforth in the description below, and in part will be apparent from thedescription, or may be learned by practice of the subject technology.The advantages of the subject technology will be realized and attainedby the structure particularly pointed out in the written description andclaims hereof as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the subject technology asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide furtherunderstanding of the subject technology and are incorporated in andconstitute a part of this specification, illustrate aspects of thesubject technology and together with the description serve to explainthe principles of the subject technology. Like reference numbersindicate like elements.

FIG. 1 is a schematic view of an exemplary CO₂-based system of thesubject technology.

FIG. 2 is a schematic view of an exemplary injection valve for theCO₂-based system shown in FIG. 1.

FIG. 3 is an exemplary diagram showing a software timing mode fordeveloping an injection sequence in the CO₂-based system of the subjecttechnology.

FIG. 4 is a gas chromatography analysis of PAs using a GC/MS with aHP5-MS, 30 m×0.32 mm, 1.0 μm film column.

FIG. 5 is a liquid chromatography analysis of PAs using an LC systemwith a BEH Phenyl 1.7 μm, 2.1×100 mm column, and UV detection (at 220nm).

FIG. 6 is a chromatographic analysis of polymer additives according tothe CO₂-based system and method of the subject technology. In generatingthe chromatogram shown in this figure, the system included a BEH 2-EP,3.0 mm×100 mm, 1.7 μm column (Waters Corp., Milford, Ma.). Peaks weredetected via UV detection (at 220 nm).

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are setforth to provide a full understanding of the subject technology. It willbe apparent, however, to one ordinarily skilled in the art that thesubject technology may be practiced without some of these specificdetails. In other instances, well-known structures and techniques havenot been shown in detail so as not to obscure the subject technology.

To facilitate an understanding of the present subject technology, anumber of terms and phrases are defined below:

Definitions

A phrase such as “an embodiment” does not imply that such embodiment isessential to the subject technology or that such embodiment applies toall configurations of the subject technology. A disclosure relating toan embodiment may apply to all embodiments, or one or more embodiments.An embodiment may provide one or more examples of the disclosure. Aphrase such “an embodiment” may refer to one or more embodiments andvice versa. A phrase such as “a configuration” does not imply that suchconfiguration is essential to the subject technology or that suchconfiguration applies to all configurations of the subject technology. Adisclosure relating to a configuration may apply to all configurations,or one or more configurations. A configuration may provide one or moreexamples of the disclosure. A phrase such as “a configuration” may referto one or more configurations and vice versa.

Furthermore, to the extent that the term “include,” “have,” or the likeis used in the description or the claims, such term is intended to beinclusive in a manner similar to the term “comprise” as “comprise” isinterpreted when employed as a transitional word in a claim.

The word “exemplary” is used herein to mean “serving as an example,instance, or illustration.” Any embodiment described herein as“exemplary” is not necessarily to be construed as preferred oradvantageous over other embodiments.

A reference to an element in the singular is not intended to mean “oneand only one” unless specifically stated, but rather “one or more.”Underlined, bold and/or italicized headings and subheadings are used forconvenience only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. All structural and functional equivalents to theelements of the various configurations described throughout thisdisclosure that are known or later come to be known to those of ordinaryskill in the art are expressly incorporated herein by reference andintended to be encompassed by the subject technology. Moreover, nothingdisclosed herein is intended to be dedicated to the public regardless ofwhether such disclosure is explicitly recited in the above description.

Unless otherwise indicated, all numbers expressing quantities such asflow volume or flow rate and so forth as used in the specification andclaims are to be understood as being modified in all instances by theterm “about.” The term “about” as used herein in reference toquantitative measurements not including the measurement of the mass of acompound, refers to the indicated value plus or minus 10%.

As used herein, the term “analyte” refers to a compound or a mixture ofcompounds (i.e., a PA or a mixture of PAs) whose analytical levels orpresence of absence in a sample is to be determined by the method or thesystem of the subject technology. PAs are a large class of compoundsthat are used in packaging materials such as high density polypropylenepill bottle (HDPE), low density polypropylene bottle (LDPE), ethylenevinyl-acetate plasma bag (EVA) and polyvinyl chloride blister pack(PVC). PAs are leachable or extractable from the packaging materials.

As used herein, the term “extractable” refers to any chemical thatmigrates from a packaging or device component under forced conditionssuch as, for example, extraction methods.

As used herein, the term “leachable” refers to any chemical migratesinto a product under normal storage conditions such as, for example,normal storage conditions.

As used herein, the term “sample” or “extract” refers to a materialwhich one desires to test for the analytical levels or the presence orabsence of the analytes, i.e., PAs. A sample may be obtained from apackaging material using conventional extraction or leaching methodsknown in the art.

As used herein, the term “hybrid”, as in “organic-inorganic hybridmaterial,” includes inorganic-based structures wherein an organicfunctionality is integral to both the internal or “skeletal” inorganicstructure as well as the hybrid material surface. The inorganic portionof the hybrid material can be, e.g., silicon, aluminum, titanium,cerium, zirconium, barium, cobalt, copper, europium, gadolinium, iron,nickel, samarium, silver, titanium, diamond, zinc, boron or oxidesthereof, silicon carbide, carbon black, carbon nanotubes, ceramic,glass, metallic materials or mixtures thereof. “Hybrid” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. The organic functionality includes organicfunctional groups which impart a certain chromatographic functionalityto a stationary phase. Exemplary organic functional groups aresubstituted or unsubstituted aliphatic groups, alicyclic groups,heterocyclic groups, aromatic groups, amino groups and the like.Exemplary hybrid materials or particles are further described in U.S.Pat. Nos. 4,017,528; 6,528,167; 6,686,035 and 7,175,913; each of whichis hereby incorporated herein by reference.

The term “aliphatic group” includes organic compounds characterized bystraight or branched chains, typically having between 1 and 22 carbonatoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynylgroups. In complex structures, the chains can be branched orcross-linked. Alkyl groups include saturated hydrocarbons having one ormore carbon atoms, including straight-chain alkyl groups andbranched-chain alkyl groups. Such hydrocarbon moieties may besubstituted on one or more carbons with, for example, a halogen, ahydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio,or a nitro group. Unless the number of carbons is otherwise specified,“lower aliphatic” as used herein means an aliphatic group, as definedabove (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having fromone to six carbon atoms. Representative of such lower aliphatic groups,e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl,3-thiopentyl, and the like. As used herein, the term “nitro” means —NO₂;the term “halogen” designates —F, —CI, —Br or —I; the term “thiol” meansSH; and the term “hydroxyl” means —OH. The term “alkylamino” as usedherein means an alkyl group, as defined above, having an amino groupattached thereto. Suitable alkylamino groups include groups having 1 toabout 12 carbon atoms, or from 1 to about 6 carbon atoms. The term“alkylthio” refers to an alkyl group, as defined above, having asulfhydryl group attached thereto. Suitable alkylthio groups includegroups having 1 to about 12 carbon atoms, or from 1 to about 6 carbonatoms. The term “alkylcarboxyl” as used herein means an alkyl group, asdefined above, having a carboxyl group attached thereto. The term“alkoxy” as used herein means an alkyl group, as defined above, havingan oxygen atom attached thereto. Representative alkoxy groups includegroups having 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms,e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms“alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogousto alkyls, but which contain at least one double or triple bondrespectively. Suitable alkenyl and alkynyl groups include groups having2 to about 12 carbon atoms, or from 1 to about 6 carbon atoms.

The term “alicyclic group” includes closed ring structures of three ormore carbon atoms. Alicyclic groups include cycloparaffins or naphtheneswhich are saturated cyclic hydrocarbons, cycloolefins which areunsaturated with two or more double bonds, and cycloacetylenes whichhave a triple bond. They do not include aromatic groups. Examples ofcycloparaffins include cyclopropane, cyclohexane, and cyclopentane.Examples of cycloolefins include cyclopentadiene and cyclooctatetraene.Alicyclic groups also include fused ring structures and substitutedalicyclic groups such as alkyl substituted alicyclic groups. In theinstance of the alicyclics such substituents can further comprise alower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a loweralkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, orthe like.

The term “heterocyclic group” includes closed ring structures in whichone or more of the atoms in the ring is an element other than carbon,for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can besaturated or unsaturated and heterocyclic groups such as pyrrole andfuran can have aromatic character. They include fused ring structuressuch as quinoline and isoquinoline. Other examples of heterocyclicgroups include pyridine and purine. Heterocyclic groups can also besubstituted at one or more constituent atoms with, for example, ahalogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, —CN, or the like. Suitable heteroaromatic andheteroalicyclic groups generally will have 1 to 3 separate or fusedrings with 3 to about 8 members per ring and one or more N, O or Satoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl,furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolidinyl.

The term “aromatic group” includes unsaturated cyclic hydrocarbonscontaining one or more rings. Aromatic groups include 5- and 6-memberedsingle-ring groups which may include from zero to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like. The aromatic ring may be substituted at one ormore ring positions with, for example, a halogen, a lower alkyl, a loweralkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a loweralkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g.,C1-C30 for straight chain or C3-C30 for branched chain. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone, e.g., C1-C20 for straight chain or C3-C20for branched chain, or 18 or fewer. In some embodiments, the cycloalkylshave from 4-10 carbon atoms in their ring structure, and more or have4-7 carbon atoms in the ring structure. The term “lower alkyl” refers toalkyl groups having from 1 to 6 carbons in the chain, and to cycloalkylshaving from 3 to 6 carbons in the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe specification and claims includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18carbon ring atoms, e.g., phenylmethyl (benzyl).

The term “aryl” includes 5- and 6-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example,unsubstituted or substituted benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine, and the like. Aryl groups also includepolycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl,and the like. The aromatic ring can be substituted at one or more ringpositions with such substituents, e.g., as described above for alkylgroups. Suitable aryl groups include unsubstituted and substitutedphenyl groups. The term “aryloxy” as used herein means an aryl group, asdefined above, having an oxygen atom attached thereto. The term“aralkoxy” as used herein means an aralkyl group, as defined above,having an oxygen atom attached thereto. Suitable aralkoxy groups have 1to 3 separate or fused rings and from 6 to about 18 carbon ring atoms,e.g., O-benzyl.

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NRaRb, in which Ra and Rb are eachindependently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb,taken together with the nitrogen atom to which they are attached, form acyclic moiety having from 3 to 8 atoms in the ring. Thus, the term“amino” includes cyclic amino moieties such as piperidinyl orpyrrolidinyl groups, unless otherwise stated. An “amino-substitutedamino group” refers to an amino group in which at least one of Ra andRb, is further substituted with an amino group.

As used herein, the term “polar surface functionality,” refers to one ormore polar functional groups or moieties that are present on the surfaceof stationary phase particles, which impart polarity on the surface ofthe particles and permit them to interact with polar analytes ormolecules. Exemplary polar functional groups include hydroxyl, aldehyde;amine; alcohol; ester; ketone; acids; acid anhydrides; metal salts;heteroatoms such as nitrogen, oxygen, the halogens, boron, phosphorus orsulphur; carbonyl, imine, oxime, N-oxide, diol, carboxy, nitrile, azide,diazonium, isonitrile, cyanate, isocyanate, or the sulphur analogues ofthe aforementioned O-containing groups. In this context, the polarfunctional group or moiety is bonded directly to surface of theparticles through the inorganic structure of the inorganic particles orthrough the organic or inorganic portions of the hybrid particles. Forexample, the direct binding of the polar functional groups to theinorganic structure of the inorganic particles or to the inorganicportion of the hybrid particles may be through the modified silane orsilanol monomers or through a carbon-silicon bond. In these particles,one or more of the functional groups (e.g., silanol) may be capped ornot.

Exemplary particles with polar surface functionality are cyano-bondedparticles in which the group bound to the surface containing acyanoalkyl group (e.g. —(CH₂)n-CN); diol-bonded particles in which thegroup bound to the surface containing a vicinal dihydroxyalkyl group(e.g., —(CH₂)n-CHOH—CH₂OH); amino-bonded particles in which the groupbound to the surface containing an aminoalkyl group (e.g., —(CH2)n-NH2);or particles with free uncapped silanol groups and no additional polargroups being bound to the surface.

As used herein, the term “polar/non-polar surface functionality,” as inparticles having a polar/non-polar surface functionality, refers toparticles that have a mixture of polar and non-polar functional groupson their surfaces. Exemplary non-polar functional groups are aliphaticgroups. For example, the aliphatic group bound to the surface of theparticles can be an alkyl chain between CI and CI8. The polar functionalgroups can be any of the ones described above. In an exemplaryembodiment, a polar/non-polar surface functionality in particles refersto a mixture of non-polar functional groups and hydroxyl groups beingpresent on the surfaces of the particles. For example, the hydroxylgroups may stem from the surface silanol groups that are uncapped.

Exemplary hybrid particles with polar/non-polar surface functionalityare alkyl-bonded particles in which the group bound to the surfacecontains an alkyl chain (usually between CI and C18); phenyl-bondedparticles in which the group bound to the surface contains a phenylgroup; or the like with the remaining functional (e.g., silanol) groupsnot being capped on the surface of the particles.

As used herein, the term “non-polar surface functionality,” as in inparticles having a non-polar surface functionality, refers to particlesthat have non-polar functional groups on their surfaces. Exemplarynon-polar functional groups are aliphatic groups. For example, thealiphatic group bound to the surface of the particles can be an alkylchain between CI and CI8. Exemplary hybrid particles with non-polarsurface functionality are alkyl-bonded particles in which the groupbound to the surface contains an alkyl chain (usually between CI andCI8); phenyl-bonded particles in which the group bound to the surfacecontains a phenyl group; or the like with the remaining functional(e.g., silanol) groups being capped on the surface of the particles.

The “capped” stationary phase (or particle) (also known as “end-capped”stationary phase or material) is a bonded stationary phase (or particle)that has been treated with a second (usually less bulky) reagent, whichis intended to react with remaining functional (e.g., silanol) groupswhich have not been substituted by the original reagent because ofsteric hindrance. Exemplary capping agents include, for example, triorganosiloxane.

As used herein, the term “calibrators” refer to preparations of PAmixtures with quantitatively known contents that are used to prepare thenecessary standards used to generate a calibration curve for PAquantification.

As used herein, the term “controls” refers to preparations of PAmixtures of known concentration, with PA concentrations representinglow, medium, and high levels of PA concentrations within the calibrationcurve range. These are used to assess the analytical batch accuracy andacceptability.

As used herein, the term “tuning mixture” refers to a mixture of PA inan appropriate solvent mixture used to optimize the performance of amass spectrometer. A tuning mixture is often a mixture of known analyteswith known concentrations that is used for tuning the massspectrometer's performance.

As used herein, the term “internal standard” refers to a labeled PA(e.g., isotopically or by fluorescence or the like) or closely relatedstructural analogs of known concentration that can be added to thesample during preparation to increase the accuracy of PA quantification.

As used herein, the term “derivatization reagents” refers to one or morecompounds that can be reacted with PAs to form a PA-complex withincreased mass spectroscopy sensitivity or improved chromatographicbehavior relative to the underivatized PA. Exemplary derivatizationmethods are alkylation, acylation and silylation, which are known in theart.

As provided above, the subject technology provides a novel method forthe analysis of PAs. In particular, the subject technology relates tothe separation and analysis of PAs using a CO₂-based chromatographysystem and method. The unique speed and resolution provided by theCO₂-based system of the subject technology allows for conducting anassay for extractables and leachables that is rapid enough to use forroutine screening. In addition, because the system does not requiregreat amounts of expensive mobile phase solvents, the cost of runningsuch assays is substantially low compared with other chromatographymethods. As discussed above, the subject technology is based, in part,on the discovery that the CO₂-based system of the present disclosureprovided a rapid separation of multiple PAs in less than about fourminutes. As shown in FIG. 6, even at such a short run time, the peaksassociates with the polymer additives were surprisingly well-resolvedwith high signal to noise ratios. Also, it was surprisingly discoveredthat the CO2-based system of the subject technology was compatible withvarious polar and non-polar extraction solvents. This compatibility willeliminate the need for solvent evaporation and sample reconstitution toinjecting the sample into the CO₂-based system of the subjecttechnology, which reduces both labor and cost. These results areattributable, in part, to the CO₂-based system of the subjecttechnology, the column chemistry and the stationary phase particle sizesused therein. Several of these attributes are discussed below.

Accordingly, in some embodiments, the subject technology relates to amethod of detecting one or more PAs in a sample comprising the steps of:providing a sample including one or more PAs; placing said sample in aCO₂-based system with one or more features described herein; subjectingthe one or more PAs to a separation column with one or more featuresdescribed herein; eluting said one or more PAs under a gradient of anorganic solvent and liquid CO₂ to form one or more eluted PAs, detectingsaid one or more PAs with a suitable detection method.

The CO₂-Based System and the Method of Use

FIG. 1 illustrates an exemplary and simplified diagram of the CO₂-basedsystem of the subject technology. As shown, the CO₂-based system 100includes a plurality of stackable modules including a solvent manager110; a system manager 140; a sample manager 170; a column manager 180;and a detector module 190.

By way of illustration and not limitation, in some embodiments, thesolvent manager 110 is comprised of a first pump 112 which receivescarbon dioxide (CO₂) from CO₂ source 102 (e.g., a tank containingcompressed CO₂). The CO₂ passes through an inlet shutoff valve 142 and afilter 144 in the system manager 140 on its way to the first pump 112.The first pump 112 can comprise one or more actuators each comprising orconnected to cooling means, such as a cooling coil and/or athermoelectric cooler, for cooling the flow of CO₂ as it passes throughthe first pump 112 to help ensure that the CO₂ fluid flow is deliverablein liquid form. In some cases, the first pump 112 comprises a primaryactuator 114 and an accumulator actuator 116. The primary andaccumulator actuators 114, 116 each include an associated pump head, andare connected in series. The accumulator actuator 116 delivers CO₂ tothe system 100. The primary actuator 114 delivers CO₂ to the system 100while refilling the accumulator actuator 116.

According to certain embodiments, the solvent manager 110 also includesa second pump 118 for receiving an organic co-solvent (e.g., methanol,etc.) from a co-solvent source 104 and delivering it to the system 110.The second pump 118 can comprise a primary actuator 120 and anaccumulator actuator 122, each including an associated pump head. Theprimary and accumulator actuators 120, 122 of the second pump 118 areconnected in series. The accumulator actuator 122 delivers co-solvent tothe system 100. The primary actuator 120 delivers co-solvent to thesystem 100 while refilling the accumulator actuator 122.

By way of illustration and not limitation, in some embodiments,transducers 124 a-d are connected to outlets of the respective pumpheads for monitoring pressure. The solvent manager 110 also includeselectrical drives for driving the primary actuators 114, 120 and theaccumulator actuators 116, 122. The CO₂ and co-solvent fluid flows aremixed at a tee 126 forming a mobile phase fluid flow that continues toan injection valve subsystem 200, which injects a sample slug forseparation into the mobile phase fluid flow.

In some embodiments, the injection valve subsystem 200 is comprised ofan auxiliary valve 220 that is disposed in the system manager 140 and aninject valve 240 that is disposed in the sample manager 170. Theauxiliary valve 220 and the inject valve 240 are fluidically connectedand the operations of these two valves are coordinated in such a manneras to reduce sample carry-over and system pressure perturbationsoccurring during injection. The reduced system pressure perturbationseliminate back flow in the column that may occur during injection and asthe result of system pressure drops. The system manager 140 includes avalve actuator for actuating the auxiliary valve 220 and electricaldrives for driving the valve actuations. Similarly, the sample manager170 includes a valve actuator for actuating the inject valve andelectrical drives for driving the valve actuations.

By way of illustration and not limitation, in some embodiments, from theinjection valve subsystem 200, the mobile phase flow containing theinjected sample slug continues through a separation column 182 in thecolumn manager 180, where the sample slug is separated into itsindividual component parts. The column manager 180 comprises a pluralityof such separation columns, and inlet and outlet switching valves 184,186 for switching between the various separation/chromatography columns.

After passing through the separation column 182, the mobile phase fluidflow continues on to a detector 192 (e.g., a flow cell/photodiode arraytype detector) housed within the detector module 190 then through a ventvalve 146 and then on to a back pressure regulator 148 in the systemmanager 140 before being exhausted to waste 106. A transducer 149 isprovided between the vent valve 146 and the back pressure regulator 148.

In some embodiments, the back pressure regulator 148 is adjustable tocontrol or modify the system fluid pressure. This can allow the pressureto be changed from run to run. The properties of CO₂ affect how quicklycompounds are extracted from the separation column 182, so the abilityto change the pressure can allow for different separation based onpressure. In certain embodiments, the back pressure regulator 148 can beused to maintain the system pressure in the range of about 1000 psi toabout 9000 psi, or in the range of about 1000 psi to about 6000 psi, orin the range of about 1000 psi to about 4000 psi, or at any particularpressure within these ranges.

By way of illustration and not limitation, in some embodiments, alsoshown schematically in FIG. 1 is a computerized system controller 108that can assist in coordinating operation of the CO₂-based system 100.Each of the individual modules 110, 140, 170, 180, 190 also includes itsown control electronics, which can interface with each other and withthe system controller 108 via an Ethernet connection 109. The controlelectronics for each module can include non-volatile memory withcomputer-readable instructions (firmware) for controlling operation ofthe respective module's components (e.g., the pumps, valves, etc.) inresponse to signals received from the system controller 108 or from theother modules. In some embodiments, each module's control electronicscan also include at least one processor for executing the computerreadable instructions, receiving input, and sending output. The controlelectronics can also include one or more digital-to-analog (D/A)converters for converting digital output from one of the processors toan analog signal for actuating an associated one of the pumps or valves(e.g., via an associated pump or valve actuator). The controlelectronics can also include one or more analog-to-digital (A/D)converters for converting an analog signal, such as from system sensors(e.g., pressure transducers), to a digital signal for input to one ofthe processors. In some embodiments, some or all of the various featuresof these control electronics can be integrated in a microcontroller.

In some embodiments, the injection valve subsystem 200 including theauxiliary valve 220 and the inject valve 240 is illustrated in FIG. 2.The auxiliary valve 220 is a rotary shear valve that includes anauxiliary valve stator 222 that has a plurality of ports, numbered 1through 6 in FIG. 2, and an auxiliary valve rotor 224 that has a rotorinterface, which includes three fluid conduits in the form of arcuategrooves 226 a-c. When assembled, the rotor interface is urged intocontact with the auxiliary valve stator 222, e.g., by pressure exertedon the auxiliary valve rotor 224 by a spring, to help ensure afluid-tight seal therebetween. The ports 1-6 are configured to receivefittings (e.g., standard compression screw/ferrule type fittings) forcoupling fluidic tubing to the auxiliary valve stator 222. In someembodiments, the auxiliary valve rotor 224 can be rotated to threediscrete angular positions, relative to the auxiliary valve stator 222,to connect the rotor grooves 226 a-c with different ones of the statorports 1-6 to form different fluidic passageways. Notably, one of thegrooves, groove 226 a, includes an extended portion 230 which allows theauxiliary valve rotor 224 to be rotated to a position in which thegroove 226 a forms a fluidic pathway between stator ports 4 and 5, whileports 1-3 and 6 are dead ended.

By way of illustration and not limitation, in some embodiments, theinject valve 240 is another six-port rotary shear valve that includes aninject valve stator 242 having a plurality of ports, numbered 1′ through6′ in FIG. 2, and an inject valve rotor 244. The inject valve rotor 244has a rotor interface, which includes three fluid conduits in the formof arcuate grooves 246 a-c. When assembled, the rotor interface is urgedinto contact with the inject valve stator 242, e.g., by pressure exertedon the inject valve rotor 244 by a spring, to help ensure a fluid-tightseal therebetween. In some embodiments, the ports 1′-6′ are configuredto receive fittings (e.g., standard compression screw/ferrule typefittings) for coupling fluidic tubing to the inject valve stator 242.Port 1′ is fluidically connected to port 4′ via a sample loop 248 (e.g.,fluidic tubing external to the inject valve stator 242). Port 2′ isfluidically connected to a metering syringe 250 and port 3′ is connectedto a needle 252. The metering syringe 250 and needle 252 are disposedwithin the sample manager 170 and are operable to aspirate sample fromvials 254 also in the sample manager 170. Port 5′ of the inject valve240 is connected to port 4 of the auxiliary valve 220, and port 6′ ofthe inject valve 240 is connected to port 1 of the auxiliary valve 220.The connections between port 2′ and the syringe 250, between port 3′ andthe needle 252, between port 5′ and port 4, and between port 6′ and port1 are made via the fluidic tubing 260 a-d.

In some embodiments, the inject valve rotor 244 can be rotated to twodiscrete angular positions, relative to the inject valve stator 242, toconnect the rotor grooves 246 a-c with different ones of the statorports 1′-6′ to form different fluid passageways.

The coordinated operation of the auxiliary and inject valves 220, 240helps to improve performance of the CO₂-based system 100 by reducing theamount of sample carry-over and can also help to reduce system pressureperturbations occurring during injection. As a result, the separationcolumn 182 may be subjected to lower pressure pulses, potentiallyincreasing the life of the column 182.

In short, during an injection, sample inside the sample loop 248 isbrought online to the fluidic tubing 260 a, 260 b connecting theauxiliary and inject valves 220, 240 while mobile phase fluid comprisinghigh pressure CO₂ flows directly from the pumps 112, 118 to theseparation column 182 via the auxiliary valve 220. The auxiliary valve220 then allows the fluidic tubing 260 a, 260 b, comprising gaseous CO₂and sample, to fill and compress with the mobile phase fluid beforeintroducing the fluid into the high pressure (e.g., about 1500 psi toabout 6000 psi) stream. The combination of these two actions can help toreduce (e.g., eliminate) carry-over anomalies and system pressure pulseswhen introducing sample into the high pressure stream. The combinationof these two actions can help to reduce (e.g., eliminate) carry-overanomalies and system pressure pulses when introducing sample into thehigh pressure stream. An exemplary process for operating the CO₂ basedsystem of the subject technology is as follows.

Step 1: Sample Manager Setup

First, the sample manager 170 (FIG. 1) sets up internally by runningvarious checks and setup procedures.

Step 2: De-Compress Sample Loop

At the start of an injection, the inject valve rotor 244 (FIG. 2) is inits inject position (from a previous injection), and the sample manager170 triggers the auxiliary valve 220 to turn its rotor 224 (60 degreescounterclockwise) to its load position. This allows the sample loop 248on the inject valve 240 and the fluidic tubing 260 a, 260 b connectingthe auxiliary and inject valves 220, 240 to vent to atmosphere. At thistime, the mobile phase fluid is permitted to flow directly from thepumps 112, 118 to the separation column 182 via the auxiliary valve 220.This pressurizes a flow path 262 (FIG. 1) between the auxiliary valve220 and the separation column 182 to a system pressure of about 1500 psito about 6000 psi.

Step 3: Aspirate Partial Loop with Needle Overfill (PLNO) Sample

Next, the sample manager 170 moves the needle 252 to a programmed vialposition, aspirates an air gap, aspirates pre-sample buffer from thevial 254, aspirates the programmed amount of sample from the vial 254,aspirates post-sample buffer from the vial 254 (see FIG. 2), and thenremoves the needle 252 from the vial and returns it toward the injectport. A final air gap is aspirated in this position. Then, the samplemanager metering syringe 250 meters the sample slug so that theinjection volume is past port 2′. The syringe 250 then dispenses 0.5 μLto take out any compliance or backlash within the system.

Step 4: Load Sample into the Sample Loop

The inject valve rotor 244 is then moved (60 degrees clockwise) to placethe inject valve 240 in its load position, with the sample loop 248 influidic communication with the meter and needle ports 2′, 3′ (FIG. 2),and the programmed sample volume is moved into the sample loop 248.

Step 5: Inject Sample into Fluidic Tubing

Within the sample manager 170, the inject valve rotor 244 is rotated (60degrees counterclockwise) to the inject position, introducing sampleinto residual gaseous CO₂ and programmed co-solvent from the previousinjection in the fluidic tubing 260 a, 260 b connecting the auxiliaryand injection valves 220, 240.

Step 6: Bring CO2 Online/Inject Sample into System

The sample manager 170 then triggers the auxiliary valve rotor 224 toturn (45 degrees clockwise) to place the auxiliary valve rotor 224 inits fill position to make the connection between ports 4 and 5 only. Atthis time, all other connections are dead ended. This action redirectsthe flow of mobile phase fluid comprising CO₂ and any programmedco-solvent from the pumps 112, 118 through the sample loop 248 and deadends against port 1 of the auxiliary valve 220. The auxiliary valverotor 224 remains in the fill position for a calculated pause time(based on mobile phase flow rate, sample loop 248 volume, and injectionvolume) until the fluidic tubing 260 a, 260 b and sample loop 248 arefilled with liquid mobile phase comprising CO₂ and any programmedco-solvent. During this time, the pressure in the flow path 262 betweenthe auxiliary valve 220 and the separation column 182 remainssubstantially at system pressure (e.g., within 500 psi) due to theresistance to flow through the separation column 182 (FIG. 1). In thisregard, the flow path 262 typically experiences a pressure drop of lessthan 500 psi while connections are dead ended.

Step 7: Inject Sample into System

The auxiliary valve rotor 224 is then rotated (an additional 15 degreesclockwise) to the inject position, completing all port connections. Thisaction redirects the flow of mobile phase comprising high pressure CO₂and any programmed co-solvent through the sample manager 170 and injectscompressed sample into the high pressure system 100.

Step 8: Wash the Needle

With the auxiliary and inject valve rotors 224, 244 in their respectiveinject positions, the sample manager 170 washes the outside and insideof the needle 252 after sample is injected. The wash syringes dispense aprogrammed amount of strong and weak washes through the inject valve 240and out through the needle 252.

By way of illustration and not limitation, FIG. 3 depicts a softwaretiming diagram used to develop the injection sequence, according to someembodiments of the subject technology. With reference to FIG. 3, thesystem controller 108 signals (402) the sample manager 170, via theEthernet connection, triggering the sample manager 170 to rotate (404)the inject valve rotor 244 to its inject position. The system controller108 also signals (406) the system manager 140 to set (408) the backpressure regulator 148 to provide the desired pressure setting. Finally,the solvent manager 110 is triggered (410) by the controller 108 to set(412) the flow and composition of the mobile phase solvent. The systemcontroller 108 waits (414) until the sample manager 170, system manager140, and solvent manager 110 have performed their respective tasks andare ready to perform a sample injection.

Then, the system controller 108 signals (416) the sample manager 170 tostart the injection sequence. In response, the sample manager 170signals (418) the solvent manager 110 to synchronize (420) the pumps(positioning plungers within the actuators in a predetermined startpoint position). The sample manager 170 then signals (422) the systemmanager 140 to move (424) the auxiliary valve rotor 224 to its loadposition. Next, the sample manager 170 executes the step of aspiratingthe PLNO sample (426), and, then, drives (428) the inject valve rotor244 to its load position. After sample is loaded (430) into the sampleloop 248, the sample manager 170 drives (432) the inject valve to theinject position. The sample manager 170 then signals (434) the solventmanager 110 to again synchronize (436) the actuator plungers.

Finally, the sample manager 170 signals (438) the system manager 140 toexecute the final movements of the auxiliary valve rotor 224. Inresponse, the system manager 140 drives (440) the auxiliary valve rotor224 to its fill position, and then pauses (442) it in the fill position(to fill and pressurize the fluidic tubing 260 a, 260 b with liquidmobile phase comprising CO₂ and programmed co-solvent). Then, the systemmanager 140 drives (444) the auxiliary valve rotor 224 to its injectposition (for injection of the sample into high pressure system).

In some embodiments, the system pressure of the CO₂-based system of thesubject technology, which is the pressure of the liquid as it exits thepump, is from about 4000 psi to about 9000 psi, or any pressurestherebetween. In an embodiment, the system pressure is any pressurebetween the ranges of about 1000 psi to about 6000 psi. In someembodiments, the system pressure controller of the CO₂-based system ofthe subject technology provides and maintains steady pressure levels,and provides accurate and reproducible pressure gradients.

In some embodiments, the pressure at the exit of the system, ascontrolled by the automated backpressure regulator (ABPR) in theCO₂-based system of the subject technology is from about 1000 psi toabout 9000 psi, or any pressures therebetween. In an embodiment, thebackpressure is any pressure between the range of about 1000 psi toabout 6000 psi. In another embodiment, the ABPR is set at 1700 psi, 2200psi, 2500 psi, 2900 psi, 3200 psi, 3500 psi. In some embodiments, theABPR of the CO₂-based system of the subject technology provides steadypressure levels and improved pressure gradients.

In some embodiments, the pre-column mobile phase dwell volume of theCO₂-based system of the subject technology is about 75 μL to about 500μL. The pre-column mobile phase dwell volume is the volume of mobilephase present in a fluidic connection or piping between a junction atwhich the CO₂ and the modifier are mixed and the head of thechromatography column. In an embodiment, the pre-column mobile phasedwell volume is about 100 μL, or about 150 μL, or about 200 μL, or about250 μL, or about 300 μL, or about 320 μL or about 350 μL, or about 400μL or about 450 μL, or any volumes therebetween.

In some embodiments, the internal diameter of the fluidic connectionthat holds the pre-column mobile phase dwell volume is about 50 μm toabout 400 μm. In some embodiments, the internal diameter of the fluidicconnection that holds the pre-column mobile phase dwell volume is about75 μm, or about 100 μm, or about 130 μm, or about 150 μm, or about 200μm, or about 250 μm, or about 300 μm, or about 350 μm, or about 375 μm,or any lengths therebetween.

In some embodiments, the post-column mobile phase dwell volume of theCO₂-based system of the subject technology is about 10 μL to about 450μL. The post-column mobile phase dwell volume is the volume of mobilephase present in a fluidic connection or piping between the end of thecolumn and the detector. In an embodiment, the post-column mobile phasedwell volume is about 10 μL, about 20 μL, about 30 μL, about 50 μL,about 90 μL, or about 120 μL, or about 150 μL, or about 200 μL, or about250 μL, or about 300 μL, or about 350 μL, or about 400 μL or any volumestherebetween.

In some embodiments, the internal diameter of the fluidic connectionthat holds the post-column mobile phase dwell volume is about is about50 μm to about 400 μm. In some embodiments, the internal diameter of thefluidic connection that holds the post-column mobile phase dwell volumeis about 75 μm, or about 100 μm, or about 130 μm, or about 150 μm, orabout 200 μm, or about 250 μm, or about 300 μm, or about 350 μm, orabout 375 μm, or any lengths therebetween.

In some embodiments, the volume of the volume of sample needed to beinjected to the CO₂-based system of the subject technology is from about0.1 μL to 20 μL, or any particular volume in between this range. Forexample, in an embodiment, the sample volume injected is 1 μL. However,those of skill in the art appreciate that the volume of sample to beinjected depends primarily on the concentration of the analytes in thatsample and also on what type of detection method being used. Forexample, if MS (Mass Spectroscopy) is the detection method used intandem with the CO₂-based system of the subject technology, smallerinjection volumes are typically required. In some embodiments, theCO₂-based system of the subject technology when in −12 tandem with anMS/MS can facilitate detection of analytes in picogram (pg, onetrillionth (10″) of a gram) ranges.

In the subject technology, the temperature fluctuations in the pumpingsystems which may result in system pressure fluctuations are reduced oreliminated, which leads to a reduced baseline noise of chromatograms ofthe CO₂-based system of the subject technology.

Alternatively or in addition, the CO₂-based system of the subjecttechnology minimizes the consumption of mobile phase solvents (e.g.methanol, acetonitrile, etc.) thereby generating less waste for disposaland reducing the cost of analysis (by more than 100 fold, in some cases)per sample.

The Column Chemistry

In various embodiments, the solid stationary phase of the chromatographycolumns of the CO₂-based system of the subject technology includesporous inorganic or inorganic/organic hybrid particles with themechanical stability and structural integrity required to withstand theoperating pressures of the system.

Inorganic particles suitable for use in the system and method of thesubject technology include silicon, aluminum, titanium, cerium,zirconium, barium, cobalt, copper, europium, gadolinium, iron, nickel,samarium, silver, titanium, diamond, zinc, boron or oxides thereof,silicon carbide, carbon black, carbon nanotubes, ceramic, glass,metallic materials or mixtures thereof. In some embodiments, suchinorganic particles may have no surface modifications. For example,without surface modifications, silica is characterized by the presenceof silanol groups on its surface. In some other embodiments, theinorganic particles, e.g., silica, may have been surface modified. Forexample, silica can be surface modified or derivatized with an organicpolar or non-polar functional group such as butyl (C₄), octyl (C₈),octadecyl (C₁₈), C₃₀, phenyl, amino, cyano, etc. A suitable commerciallyavailable column that includes such particle is, for example, the“ACQUITY UPC2 HSS C18 SB column, Waters Corporation, Milford, Ma.”

Hybrid particles suitable for use in the system and method of thesubject technology include an inorganic portion such as, e.g., alumina,silica, titanium or zirconium oxides, or ceramic material; and anorganic portion bonded to one or more atoms of the inorganic portion.Exemplary hybrid materials are disclosed in U.S. Pat. No. 4,017,528, thetext of which is incorporated herein by reference.

In some embodiments, the organic portion of the hybrid particles is aC1-C18 aliphatic or aromatic moieties (which may additionally besubstituted with alkyl, aryl, cyano, amino, hydroxyl, diol, nitro,ester, ion exchange or embedded polar functionalities) or a substitutedor unsubstituted CI-CI8 alkylene, alkenylene, alkynylene or arylenemoiety. In one embodiment where the inorganic portion is silica, “hybridsilica” refers to a material having the formula Si0₂/(R_(p) ²R_(q)⁴SiO_(t))_(n) or Si0₂/[R⁶(R_(r) ²SiO_(t))_(m)]_(n) (disclosed in U.S.Pat. Nos. 7,919,177; 7,223,473, and 246,686,035, each of which is herebyincorporated herein by reference) wherein R² and R⁴ are independentlyC₁-C₁₈ aliphatic or aromatic moieties (which may additionally besubstituted with alkyl, aryl, cyano, amino, hydroxyl, diol, nitro,ester, ion exchange or embedded polar functionalities), R6 is asubstituted or unsubstituted Q-Cig alkylene, alkenylene, alkynylene orarylene moiety bonded to one or more silicon atoms or bridging two ormore silicon atoms, p and q are 0, 1 or 2, provided that p+q=1 or 2, andthat when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, provided thatwhen r=0, t=1.5, and when r=1, t=1; m is an integer greater than orequal to 2, and n is a number from 0.03 to 1, or alternatively, 0.1 to1, or alternatively 0.2 to 0.5. R² may be additionally substituted witha functionalizing group R.

The functionalizing group R includes organic functional groups whichimpart a certain chromatographic functionality to a chromatographicstationary phase, including, e.g., octadecyl (Cig) or phenyl. Suchfunctionalizing groups are present in, e.g., surface modifiers such asdisclosed herein which are attached to the base material, e.g., viaderivatization or coating and later crosslinking, imparting the chemicalcharacter of the surface modifier to the base material. In anembodiment, such surface modifiers have the formula Z_(a)(R′)_(b)Si—R,where Z═Cl, Br, I, C1-C5 alkoxy, dialkylamino, e.g., dimethylamino, ortrifluoromethanesulfonate; a and b are each an integer from 0 to 3provided that a+b=3; R′ is a C₁-C₆ straight, cyclic or branched alkylgroup, and R is a functionalizing group. R′ may be, e.g., methyl, ethyl,propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexylor cyclohexyl; in an embodiment, R′ is methyl.

The porous inorganic/organic hybrid particles possess both organicgroups and silanol groups which may additionally be substituted orderivatized with a surface modifier. “Surface modifiers” include(typically) organic functional groups which impart a certainchromatographic functionality to a chromatographic stationary phase.Surface modifiers such as disclosed herein are attached to the basematerial, e.g., via derivatization or coating and later crosslinking,imparting the chemical character of the surface modifier to the basematerial. In one embodiment, the organic groups of the hybrid particlereact to form an organic covalent bond with a surface modifier. Thesurface modifiers can form an organic covalent bond to the particle'sorganic group via a number of mechanisms well known in organic andpolymer chemistry including but not limited to nucleophilic,electrophilic, cycloaddition, free-radical, carbene, nitrene, andcarbocation reactions. Organic covalent bonds are defined to involve theformation of a covalent bond between the common elements of organicchemistry including but not limited to hydrogen, boron, carbon,nitrogen, oxygen, silicon, phosphorus, sulfur, and the halogens. Inaddition, carbon-silicon and carbon-oxygen-silicon bonds are defined asorganic covalent bonds, whereas silicon-oxygen-silicon bonds that arenot defined as organic covalent bonds.

In some embodiments, the solid stationary phase of the separationcolumns of the subject technology includes a monolith, particles, porousparticles, and/or superficially porous particles. Particles can bespherical or non-spherical. The solid stationary phase can includesilica, inorganic silica, and/or metal oxide. In some embodiments, thecolumn is equipped with one or more frits to contain the stationaryphase material. In embodiments in which the stationary phase material ismonolithic, the housing may be used without the inclusion of one or morefrits.

The solid stationary phase includes, for example, particles having amean size within the range of about 0.5-3.5 microns, though a smaller orlarger size could be selected if appropriate for a desired application.In various embodiments, the mean particle size is about 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5microns. In general, particle size can be selected in view of thedesired pressure and/or flow rate. For example, larger particle size canbe used to achieve consistent pressure from a column head to an endduring high pressurized digestion. Alternatively, smaller particle sizesresult in higher flow rates and higher efficiency, which yield fasterand more sensitive separations. The solid stationary phase can includepores having a mean pore volume within the range of 0.1-2.5 cm/g. Invarious examples, the mean pore volume is about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 cm/g. In some embodiments, porousparticles may be advantageous because they provide a relatively largesurface area (per unit mass or column volume) for protein coverage atthe same time as the ability to withstand high pressure.

The solid stationary phase can include pores having a mean pore diameterwithin the range of 100-1000 Angstroms. For example, in someembodiments, the mean pore diameter of the solid stationary phaseparticles is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000Angstroms, or any value or range therebetween.

In certain embodiments, the chromatography or separation column of thesubject technology includes (a) a column having a cylindrical interiorfor accepting a packing material, and (b) a packed chromatographic bedcomprising a porous material comprising an organosiloxane/Si02 materialhaving the formula SiO2/(R²pR_(q) ⁴SiO_(t))n or SiO2/[R⁶(R_(r)²SiO_(t))_(m)]n, as 24 described above, wherein R² and R⁴ areindependently CI-CI 8 aliphatic, styryl, vinyl, propanol, or aromaticmoieties, R⁶ is a substituted or unsubstituted Q-Qg alkylene,alkenylene, alkynylene or arylene moiety bridging two or more siliconatoms, p and q are 0, 1 or 2, provided that p+q=1 or 2, and that whenp+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, provided that when r=0,t=1.5, and when r=1, t=1; m is an integer greater than or equal to 2,and n is a number from 0.03 to 1, said porous hybrid silicachromatographic matrix having a chromatographically-enhancing poregeometry and average pore diameters of about 100 to 300 A. In anembodiment, the porous particles of hybrid silica have been surfacemodified. In another embodiment, the particles have been surfacemodified by a surface modifier selected from the group consisting of anorganic group surface modifier, a silanol group surface modifier, apolymeric coating surface modifier, and combinations thereof. In anotherembodiment, the surface modifier has the formula Z_(a)(R′)bSi—R, whereZ═Cl, Br, I, C₁-C₅ alkoxy, dialkylamino or trifluoromethanesulfonate; aand b are each an integer from 0 to 3 provided that a+b=3; R′ is a C₁-C₆straight, cyclic or branched alkyl group, and R is a functionalizinggroup.

The functionalizing group R may include alkyl, alkenyl, alkynyl, aryl,cyano, amino, diol, nitro, cation or anion exchange groups, or alkyl oraryl groups with embedded polar functionalities. Examples of suitable Rfunctionalizing groups include C1-C30 alkyl, including C₁-C₂₀, such asoctyl (C₈), octadecyl (C₁₈), and triacontyl (C₃₀); alkaryl, e.g.,C₁-C₄-phenyl; cyanoalkyl groups, e.g., cyanopropyl; diol groups, e.g.,propyldiol; amino groups, e.g., aminopropyl; and alkyl or aryl groupswith embedded polar functionalities, e.g., carbamate functionalitiessuch as disclosed in U.S. Pat. No. 5,374,755, the text of which isincorporated herein by reference. In an embodiment, the surface modifieris an organotrihalosilane, such as octyltrichlorosilane oroctadecyltrichlorosilane. In another embodiment, the surface modifiermay be a halopolyorganosilane, such as octyldimethylchlorosilane oroctadecyldimethylchloro silane.

In another embodiment, the hybrid particle's organic groups and silanolgroups are both surface modified or derivatized. In another embodiment,the particles are surface modified by coating with a polymer. In certainembodiments, surface modification by coating with a polymer is used inconjunction with silanol group modification, organic group modification,or both silanol and organic group modification.

Polymer coatings are known in the literature and may be providedgenerally by polymerization or polycondensation of physisorbed monomersonto the surface without chemical bonding of the polymer layer to thesupport (type I), polymerization or polycondensation of physisorbedmonomers onto the surface with chemical bonding of the polymer layer tothe support (type II), immobilization of physisorbed prepolymers to thesupport (type III), and chemisorption of presynthesized polymers ontothe surface of the support (type IV). See, e.g., Hanson et al., J.Chromat. A656 (1993) 369-380, the text of which is incorporated hereinby reference. As noted above, coating the hybrid material with a polymermay be used in conjunction with various surface modifications describedin U.S. Pat. Nos. 7,919,177; 7,223,473, and 6,686,035, each of which ishereby incorporated herein by reference. Additional inorganic/organichybrid particles are disclosed in WO2010141426, which is herebyincorporated herein by reference.

Exemplary suitable commercially available columns that include suchinorganic/organic hybrid particles include, for example, the “ACQUITYUPC² ethylene bridged hybrid (BEH), BEH 2-EP, and charged surface hybrid(CSH) CI8 SB columns, Waters Corporation, Milford, Ma.”

In one exemplary embodiment, the particles used in the separationcolumns of the CO₂-based system of the subject technology have thefollowing specifications:

Particle Pore Surface Carbon Particle Size Size Area Load End- ChemistryShape (μm) (Å) (m²/g) (%) capped Hybrid particles Spher- 1.7, 3.5 135185 9 No with a polar surface ical functionality (e.g., BEH 2-ethylpridine) Hybrid particles Spher- 1.7, 3.5 135 185 N/A N/A withsurface silanol ical groups but no additional surface functionality(e.g., BEH) Hybrid particles Spher- 1.7, 3.5 135 185 10 No with surfaceical modification/ polymer coating (e.g., CSH Flouro-phenyl) Inorganicsilica Spher- 1.7, 3.5 100 230 8 No particles with a ical surfacefunctionality (e.g., HSS C₁₈ SB)

In some embodiments, the depending on the complexity and nature of thesample components, the separation is accomplished using a hybridmaterial stationary phase modified with an alternate ligand (polar,non-polar, or ionic), or one with no additional surface modification atall. Additionally, the separation could be achieved on various particlessizes below 5 μm in diameter. In some embodiments, the internal diameter(ID) of the chromatography column of the subject technology is betweenabout 1 mm to 5 mm, or between about 2 mm to 4 mm. In an embodiment, theID of the column is about 3 mm. In some embodiments, the length of thechromatography column of the subject technology is between about 30 mmto 200 mm or between about 50 mm to 150 mm. In an embodiment, the lengthof the chromatography column is 50 mm. In another embodiment, the lengthof the chromatography column is 150 mm.

In some embodiments, depending on the column dimension chosen andoptimization necessary, the flow rate of the mobile phase is set betweenabout 0.1 mL/min to 4 mL/min, or any intervals therebetween, e.g.,between about 0.5 mL/min to 3.5 mL/min, with a backpressure regulatorsetting of about 1000-9000 psi or about 1000-4000 psi. In otherembodiments, the temperature at which the chromatography column operatesis adjusted to optimize the analyte separations with a practical workingrange of about 5° C. to 85° C., or any specific temperature within thisrange. In some embodiments, the column compartment temperature rangesfrom about 40° C. to 70° C. In one embodiment, the column compartmenttemperature ranges from about 20° C. to 70° C. In another embodiment,the column compartment temperature is kept at about 20° C. or at about85° C. or at any specific temperature between about 5° C. to 85° C.

The Mobile Phase Solvent

In some embodiments, the method of the subject technology relates tomethod of detecting the presence or absence or levels of an analyte or asolute (i.e., a PA or a mixture of extractable and/or leachable PAs) ina mixture. Thus, according to certain embodiments of the subjecttechnology, a solution having an analyte is contacted with a porousmaterial of the separation column under conditions that allow forsorption of the analyte to the porous material. The analyte can be,e.g., any molecule having a hydrophobic, hydrophilic, or ionicinteraction or a combination of two or three of these interactions. Theporous material having the sorbed the analyte is eluted with a solventunder conditions so as to desorb the analyte from the porous material.The level of the desorbed solute (e.g., eluted PAs) present in thesolvent eluted from the separation column after the elution can then bedetected using a suitable detection method.

In general, liquid CO₂ is used as the main mobile phase solvent of thesubject technology to desorb the solute(s). In some embodiments, theliquid CO₂ is in a supercritical state. In some embodiments, the liquidCO₂ is in a subcritical state. In some embodiments, the physical stateof the liquid CO₂ changes between supercritical and subcritical or viceversa. Due to its miscibility, the CO₂ solvent can be combined with oneor more modifiers (co-solvents) for more effective desorption or elutionof the analytes from the chromatography column.

In some embodiments, suitable modifiers to be combined with the CO₂mobile phase include, e.g., polar water-miscible organic solvents, suchas alcohols, e.g., methanol, ethanol or isopropanol, acetonitrile,acetone, and tetrahydrofuran, or mixtures of water and any of thesesolvents. In other embodiments, the modifiers include, e.g., nonpolar ormoderately polar water-immiscible solvents such as pentane, hexane,heptane, xylene, toluene, dichloromethane, diethylether, chloroform,acetone, dioxane, THF, MTBE, ethylacetate or DMSO. Mixtures of thesemodifiers are also suitable. In some embodiments, modifiers or modifiermixtures must be determined for each individual case. A suitablemodifier can be determined by one of ordinary skill in the art withoutundue experimentation, as is routinely done in chromatographic methodsdevelopment.

In one embodiment, the ratio of a modifier to CO₂ (v/v) is between about0.0001 to 1 to about 1 to 1. In another embodiment, this ratio ofmodifier to CO₂ (v/v) is between about 0.001 to 1 to about 1 to 1, orany ratios in between. In certain embodiments, the amount of themodifier added to CO₂ is constant or changes in a gradient mode(increasing or decreasing), or is a combination of both, during theelution period. In certain other embodiments, the modifier is added tothe CO₂ mobile phase at a constant rate of, for example, 8%, or 10%, or20%, or 25% over the elution period.

In some embodiments the modifier is added, in an increasing gradientmode, from about 0% to 50% (v/v to CO₂), or from about 8% to 33%, orfrom about 6% to 35%, or from about 4% to 37%, or from about 9% to 40%(v/v to CO₂), or from about 8% to 27%, or from about 11%-30%, or anyother intervals within the 0% to 50% (v/v to CO₂) range, over theelution period.

In some embodiments the modifier is added, in a decreasing gradientmode, from about 50% to 0% (v/v to CO₂), or from about 33% to 8%, orfrom about 35% to 6%, or from about 37% to 4%, or from about 40% to 9%(v/v to CO₂), or from about 27% to 8%, or from about 30%-11%, or anyother intervals within the 50% to 0% (v/v to CO₂) range, over theelution period. In some embodiments, the modifier is added to the CO₂mobile phase in a gradient of 0% to about 50% (v/v CO₂) (or anyascending percentage range within 0% to 50%) in about 2 to 4 min (or anyfraction of time within this range) with a hold period at a constantmodifier percentage at the beginning, at the end or at any time duringthe elution period. For example, in an embodiment, the hold period isfor about 0.1 to 3 min (or any fraction of time within this range) atconstant modifier volume of, e.g., 5%, 10%, 15%, 20%, 30%, 40% (v/v toCO₂) or more. In some embodiments, the modifier is added in gradients of0% to about 70% or less (v/v CO₂), 0% to about 50% or less (v/v to CO₂),or 0% to about 30% or less (v/v to CO₂) over the elution period.

In some embodiments, the modifier is added to the CO₂ mobile phase in agradient of about 50% to 0% (v/v CO₂) (or any descending percentagevalue within 50% to 0%) in about 2 to 4 min (or any fraction of timewithin this range) with a hold period at a constant modifier percentageat the beginning, end or anytime during the elution period. In someembodiments, the modifier can be added in gradient of about 70% to 0% ormore (v/v CO₂), about 50% to 0% or more (v/v CO₂), or about 30% to 0% ormore (v/v CO₂). In an embodiment, the modifier is added to CO2 with agradient of 0% to about 25% in 2.5 min. and a hold at 25% for 1 minute.

In some embodiments, depending on the column dimension chosen andoptimization necessary, the flow rate of the mobile phase is set betweenabout 0.1 mL/min to 4 mL/min during the elution period. In anembodiment, the mobile phase flow rate increases in a gradient of about0.5 mL/min to 4.0 mL/min, or any intervals therebetween. In anotherembodiment, the mobile phase flow rate decreases in a gradient of 4.0mL/min to 0.5 mL/min, or any intervals therebetween. In anotherembodiment, the flow rate remains constant at, for example, about 0.8mL/min or about 2 mL/min or about 3.5 mL/min.

In some embodiments, depending on the nature of the PA being detected ortested, the mobile phase further includes one or more additives foroptimizing the separation. In an embodiment, one or more additivesincluding, e.g. formic acid, ammonium acetate, isopropyl amine, diethlyamine, ammonium hydroxide or the like, are added to the mobile phase ata concentration range of about 0.5% to 5% (v/v to modifier) or anyspecific percentage within this range. In an embodiment, the additive isadded at a constant amount of 3% (v/v modifier) over the elution period.

In another embodiment, depending on the polarity of the PA beingdetected or tested, the gradient duration (tg) of the mobile phase isvaried between about 0.1 min to 12 min or any specific period withinthis range. In an embodiment, tg is about 1 min, or about 2 min, orabout 3 min, or about 5 min, or about 7 min. In some embodiments, theentire elution period is less than or equal to about 12 min, or less orequal to than about 8 min, or less than or equal to about 5 min, or lessthan or equal to about 4 min, or less than or equal to about 3 min, orless than or equal to about 2 min, or less than or equal to about 1.5min.

Due to the reason that supercritical and/or liquid CO₂ is miscible withthe entire eluotropic series, various polar and non-polar modifiers canbe added to CO₂ to facilitate desorption of a wide variety of analytes.A related advantage of the CO₂-based system of the subject technology isits compatibility with a wide range of sample solutions and solvents.Since liquid CO₂ is miscible with a wide range of solvents, a sampleprepared for analysis by the method of the subject technology does nothave to be subject to a solvent exchange step prior to analysis by themethod of subject technology.

In some embodiments, suitable detection methods for detecting thesolutes, include, but not limited to, UV, PDA, Evaporative LightScattering (ELS), CD, FID and Mass Spectroscopy (MS).

Sample Preparation

A sample for analysis can be any sample from biological ornon-biological sources that may contain PAs or is suspected of havingPAs. For example, a sample can be a piece of packaging material formedicines (e.g., solid, semi-solid or liquid dosage form), food (e.g.,meat, dairy, or vegetative sample) or beverages (e.g., soft drinks,orange juice or milk).

A sample can be treated to remove components that could interfere withthe detection techniques such as a mass spectrometry technique. Avariety of extraction and purification techniques known to those havingskill in the art can be used based on the sample type. Solid samples canbe grinded and extracted to free the analytes of interest frominterfering components. In such cases, a sample can be centrifuged,filtered, and/or subjected to chromatographic techniques to removeinterfering components (e.g., cells or tissue fragments). In yet othercases, reagents known to precipitate or bind the interfering componentscan be added.

In some embodiments, following a sample preparation step (e.g.,purification, extraction), the sample is dissolved in a diluentcontaining at least about 60% organic solvent or at least about 70%organic solvent, or at least about 80% organic solvent, or at leastabout 90% organic solvent. In other embodiments, following a samplepreparation step, the sample includes a polar or non-polar organicsolvent, a mixture of organic solvents, or a mixture of water or anaqueous solution and a water-miscible polar organic solvent, e.g.,methanol, ethanol, isopropanol, tetrahydrofuran, dicholormethane,hexane, N,N-dimethylformamide, dimethylsulfoxide, acetonitrile or acombination thereof. In an embodiment, the solution is an acidic, basicor neutral solution having between about 1% and about 99% water byvolume. In another embodiment, the sample solution is an acidic, basicor neutral solution having between about 1% and about 75% water byvolume. In another embodiment, the sample solution comprises a non-polarorganic solvent such as, for example, hexane. The sample solutioncomprising the analyte can, optionally, further contain one or moreadditional solutes. In one embodiment, the sample solution is an aqueoussolution which includes a complex variety of analytes and solutes. Asample solution may be extracts of packaging materials or extracts ofmedical devices that come in contact with food, drugs, or the humanbody. Such extracts can be, e.g., aqueous extracts or organic extractswhich have been dried and subsequently reconstituted in water or in awater/organic mixture. Such extracts can also be, e.g., aqueous extractsor organic extracts which have not been dried or subsequentlyreconstituted in water or in another solvent. The solution can becontacted with the porous material in any fashion which allows sorptionof the solute to the porous material, such as a batch or chromatographicprocess. In some embodiments, no derivatization step of the solutes isnecessary as the CO₂-based system of the subject technology iscompatible with a wide range of sample solutions.

In one embodiment, the extracted or purified sample which may include anaqueous or organic solvent or diluent is dried and subsequentlyreconstituted in a solvent (e.g., water or water/organic mixture) thatis compatible with the mobile phase of the method and system of thesubject technology. This is known as a solvent exchange step. Forexample, the extracted or purified sample is dried and thenreconstituted in methanol or in methanol and water.

In another embodiment, the extracted or purified sample which isdissolved in an aqueous or organic solvent or in a diluent with at least60% organic solvent will not undergo solvent exchange or will not besubject to a solvent exchange step before being analyzed by the methodand/or system of the subject technology. The absence of a solventexchange step shortens the analysis period and improves the run time ofthe method of subject technology.

In some embodiments, sample is prepared with or extracted in an organicsolvent wherein the prepared sample is analyzed by the method of thesubject technology without additional sample derivatization or solventexchange (i.e., drying of the solvent and reconstituting the analytes orsolutes with a different solvent). The absence of a derivatization stepshortens the analysis period and improves the run time of the method ofsubject technology. In certain embodiments, the analytes are derivatizedbefore analysis by the method and/or system of the subject technology.Various methods for derivatizing analytes are known in the art. Ingeneral derivatization fall into three general reaction types: (1)Alkylation of which the general process is esterification, (2) Acylationand (3) Silylation. Common derivatization reagents for the Alkylationtype of reactions are Dialkylacetals, Diazoalkales, Pentafluorobenzylbromide (PFBBr), Benzylbromide, Boron trifluoride (BF3) in methanol orbutanol and Tetrabutylammonium hydroxide (TBH) among others. Reagentsused for the silylation derivatization process includeHexamethyldisilzane (HMDS), Trimethylchlorosilane (TMCS),Trimethylsilylimidazole (TMSI), Bistrimethylsilylacetamide (BSA),Bistrimethylsilyltrifluoroacetamide (BSTFA),N-methyltrimethylsilyltrifluoroacetamide (MSTFA),Trimethylsilyldiethylamine (TMS-DEA),Nmethyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA), andHalo-methylsilyl derivatization reagents. Common reagents for theAlkylation process are Fluoracylimidazoles, Fluorinated Anhydrides,N-Methyl-bis(trifluoroacetamide) (MBTFA), Pentafluorobenzoyl Chloride(PFBCI) and Pentafluoropropanol (PFPOH).

In certain embodiments, an internal standard can be added to a sampleprior to sample preparation. Internal standards can be useful to monitorextraction/purification efficiency. An internal standard can be added toa sample and allowed to equilibrate for a period of time, e.g., 5, 10,15, 20, 25, 30, 60, 120 or more minutes. Equilibration temperature canbe from about 10° C. to about 45° C., or any value in between (e.g., 15,25, 30, 35, 37, 42, or 44° C.). In certain cases, equilibration can beat room temperature for about 15 minutes.

An internal standard can be any compound that would be expected tobehave under the sample preparation conditions in a manner similar tothat of one or more of the analytes of interest. For example, astable-isotope-labeled version of an analyte of interest can be used,such as a deuterated version of an analyte of interest. While not beingbound by any theory, the physicochemical behavior of suchstable-isotope-labeled compounds with respect to sample preparation andsignal generation would be expected to be identical to that of theunlabeled analyte, but clearly differentiable by mass on a massspectrometer.

To improve the run time and minimize hands-on sample preparation,on-line extraction and/or analytical chromatography of a sample can beused. For example, in certain methods, a sample, such as a urine orblood sample can be extracted using an extraction column, followed byelution onto an analytical chromatography column. The columns can beuseful to remove interfering components as well as reagents used inearlier sample preparation steps (e.g., to remove reagents such asacetonitrile). Systems can be coordinated to allow the extraction columnto be running while an analytical column is being flushed and/orequilibrated with solvent mobile phase, and vice-versa, thus improvingefficiency and run-time. A variety of extraction and analytical columnswith appropriate solvent mobile phases and gradients can be chosen bythose having ordinary skill in the art.

Various extraction methods are known in the art that can be used toprepare a sample before it being analyzed by the subject technology.Such extraction methods include, but are not limited to, sonication,soxhlet extraction, microwave assisted extraction (MAE), supercriticalfluid extraction (SFE), accelerated solvent extraction (ASE),pressurized liquid extraction (PLE), pressurized hot water extraction(PHWE) and/or surfactant assisted extraction (PHWE) in common solventssuch as methanol, ethanol, mixtures of alcohols and water, or organicsolvents such as ethyl acetate or hexane.

In some embodiments, the concentration of the PAs in the sample solutionis about 5 mg/mL, 4, mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.1 mg/mL, 0.05mg/mL, 0.01 mg/mL, 0.005 mg/mL, 0.001 mg/mL, 0.0001 mg/mL, 1×10⁻⁵ mg/mL,1×10⁻⁶ mg/mL or less. In an embodiment, the concentration of the PAs inthe sample solution being analyzed by the method and system of thesubject technology is in ng/mL or pg/mL range or lower. In anotherembodiment, the sample injection volume for injection into the CO₂ basedsystem of the subject technology is about 10 μL, 8 μL, 6 μL, 5 μL, 4 μL,3 μL, 2 μL, 1 μL.

Detection

In some embodiments, suitable detection methods for detecting theanalytes include, but not limited to, UV, photodiode array (PDA),Evaporative Light Scattering (ELS), CD, FID, and Mass Spectrometry (MS).

Depending on the sample that is being analyzed by the method and systemof the subject technology, a suitable detector may be used. Suitabledetectors are known in the art. For example, if the sample is containsan abundance of analytes (i.e., about 1-10 ppm range or 1 to 10 μg/mL),a detection method such as UV, PDA or ELS may be used. If the samplecontains a minute amount of analytes (in ng/mL or pg/mL range), adetection method such as Mass Spectrometry may be used.

Kits

One embodiment of the subject technology features a kit for performingthe method of the subject technology. As used herein, the term kitrefers to a collection of parts and reagents bundled together withsuitable packaging and instructions for their use. One kit forperforming an analysis of a sample for the analytical levels or thepresence or absence of PAs, in accordance with the subject technologyincludes internal standards for calibrating and facilitating theidentification of one or more PAs; sample preparation devices orreagents and materials for performing sample purification, extraction,preparation or derivatization; and a chromatography column forseparating or detecting PAs of the sample.

The foregoing description is provided to enable a person skilled in theart to practice the various configurations described herein. While thesubject technology has been particularly described with reference to thevarious figures and configurations, it should be understood that theseare for illustration purposes only and should not be taken as limitingthe scope of the subject technology.

There may be many other ways to implement the subject technology.Various functions and elements described herein may be partitioneddifferently from those shown without departing from the scope of thesubject technology. Various modifications to these configurations willbe readily apparent to those skilled in the art, and generic principlesdefined herein may be applied to other configurations. Thus, manychanges and modifications may be made to the subject technology, by onehaving ordinary skill in the art, without departing from the scope ofthe subject technology.

It is understood that the specific order or hierarchy of steps in theprocesses disclosed is an illustration of exemplary approaches. Basedupon design preferences, it is understood that the specific order orhierarchy of steps in the processes may be rearranged. Some of the stepsmay be performed simultaneously. The accompanying method claims presentelements of the various steps in a sample order, and are not meant to belimited to the specific order or hierarchy presented.

This subject technology is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, are incorporated herein by reference.

EXAMPLES Example 1 Sample Preparation for Chromatographic Analysis ofPAs

Four different types of packaging material were extracted: high densitypolypropylene pill bottle (HDPE), low density polypropylene bottle(LDPE), ethylene vinyl-acetate plasma bag (EVA) and polyvinyl chlorideblister pack (PVC). Extraction techniques used for sample preparationinvolved microwave, Soxhlet, and supercritical fluid extraction (SFE).The extracts were rapidly screened for 14 common polymer additives.

Microwave Sample Preparation:

For hexane and isopropanol samples, 2 g of plastic were cut into 1×1 cmpieces and extracted in 10 mL of solvent for 3 h at 50° C.

Water Samples:

Water samples were prepared in a conventional oven. Two grams of samplein 10 mL of water were kept for 72 h at 50° C.

Soxhlet Sample Preparation:

Soxhlet extractions were carried out by placing cut pieces (1×1 cm) ofmaterial (3 g for PVC, 5 g for HDPE, LDPE, and EVA) into a Whatman 33mm×94 mm cellulose extraction thimble. Approximately 175 ml ofextraction solvent (either hexane or IPA) was added into the Soxhletapparatus.

All samples were extracted with the hot boiling solvent mixture for 8hours. Upon completion, the extraction solvent was reduced to neardryness and reconstituted in 15 mL of either hexane or IPA(isopropanol).

SFE Sample Preparation:

Supercritical fluid extraction (SFE) was performed using a Waters MV-10ASFE™ System. For each SFE experiment, cut pieces (roughly 1×1 cm) ofmaterial were loaded into 10 mL stainless steel extraction vessels (2 gfor PVC, 3 g for HDPE, LDPE, and EVA). Two distinct extractions wereperformed on each material, the first used 5 mL/min carbon dioxide plus0.10 mL/min IPA (low IPA), the second used 4 mL/min carbon dioxide plus1.0 mL/min IPA (high IPA). All extractions were performed at 50° C. and300 BAR back pressure using a 30 minute dynamic, 20 minute static, and10 minute dynamic program which was repeated 2 times. IPA was used as amakeup solvent at 0.25 mL/minute. Following extraction, collectedsolvent (a mixture of the co-solvent and make-up solvent) was reduced tonear dryness and reconstituted in IPA (10 mL for PVC, 9 mL for HDPE,LDPE, and EVA) for high IPA extraction. For low IPA extractions, thecollected solvent was brought up to volume accordingly.

Example 2 Gas Chromatography Analysis of Extractable and/or LeachablePAs

In this example, GC-MS was used to analyze PAs in hexane and IPAextracts of the packaging materials described in Example 1. The GC-MSmethod used in this example had the following parameters: System: WatersSynapt G2 S HDMS® with 7890A GC; Column: HP5-MS, 30 m×0.32 mm, 1.0 μmfilm; Carrier gas: He at 2 mL/min; Temperature program: 35° C. for 5min, 20° C./min to 320° C. hold 20.75 min; Injection port: 300° C.;Injection type: 1 μL splitless, 1 min purge; Makeup gas: N2 at 400mL/min; Transfer line: 350° C.; Scan range: 100-1500 Da; Run time: 40min.

As shown in FIG. 4, it took about 40 minutes for the GC-MS to generatepeaks corresponding to the analytes of the sample solutions.

Example 3 Liquid Chromatography Analysis of Extractable and/or LeachablePAs

In this example, an LC with a photodiode array detector (PDA) was usedto detect PAs in water and IPA extracts of the packaging materialsdescribed in Example 1. The LC method used in this example had thefollowing parameters: System: Waters ACQUITY UPLC® (Waters Corp.,Milford, Ma.) with PDA and SQD; Column: ACQUITY UPLC BEH Phenyl 1.7 μm,2.1×100 mm (Waters Corp., Milford, Ma.); Mobile phase A: 0.1% formicacid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Flowrate: 0.9 mL/min; Gradient: 50% B to 90% over 10 min. Reequilibrate backto 50% B; Column Temp: 50° C.; Injection volume: 2 μL; Run time: 12 min;Wavelength of the PDA detector: 220 nm.

The results are shown in FIG. 5. As shown in this figure, it took the LCinstrument about 9 minutes to resolve the peaks for PAs in the samplesolutions.

Example 4 Rapid Separation and Analysis of PAs According to the Methodand System of the Subject Technology

In this example, samples solutions from three solvent extracts, i.e.,hexane, isopropanol (IPA) and water were analyzed for PAs according tothe method and system of the subject technology. The parameters for theCO₂-based system were as follows: System: Waters ACQUITY UPC²® (WatersCorp., Milford, Ma.) with PDA and SQD; Column: ACQUITY UPC² BEH 2-EP,3.0 mm×100 mm, 1.7 pm (Waters Corp., Milford, Ma.); Modifier:Methanol/Acetonitrile (1:1); Flow rate: 2 mL/min; Gradient: 1% modifier(v/v CO₂) for 1 min, to 20% over 2.5 min, hold for 30 s, re-equilibrateback to 1% modifier; Column Temp: 65° C.; APBR: 1800 psi; InjectionVolume: 1.0 μL; Run Time: 5.1 minutes; Wavelength: 220 nm.

The results (FIG. 6) demonstrated that the subject technology provides arapid simple analytical method for detecting a wide range of PAs invarious solvents. In addition, these results showed that the system andmethod of the subject technology facilitated the generation of achromatogram with high peak resolution and high signal-to-noise ratios,which was unexpected.

While certain aspects and embodiments of the subject technology havebeen described, these have been presented by way of example only, andare not intended to limit the scope of the subject technology. Indeed,the novel methods and systems described herein may be embodied in avariety of other forms without departing from the spirit thereof. Theaccompanying claims and their equivalents are intended to cover suchforms or modifications as would fall within the scope and spirit of thesubject technology.

What is claimed is:
 1. A method for detecting one or more polymeradditives (PAs) in a sample by means of a CO₂-based chromatographyanalysis comprising: providing a sample comprising one or more PAs foranalysis; wherein the sample is prepared with, extracted or dissolved ina diluent comprising at least 60% organic solvent, with the proviso thatthe sample is analyzed without a solvent exchange step; applying thesample to a chromatography column with a solid stationary phasecomprising silica particles having silanol groups on a surface of thesilica particles and having a mean particle size of about 0.5 to about3.5 microns; eluting the one or more PAs from the chromatography columnby a mobile phase comprising a mixture of liquid CO₂ and a modifier toform one or more eluted PAs, wherein the mobile phase has dwell volumeof about 75 μL to about 500 μL; and detecting said one or more elutedPAs.
 2. The method of claim 1, wherein the sample is not subject to aderivatization step.
 3. The method of claim 1, wherein the particleshave a mean particle size of about 0.5 to about 2 microns.
 4. The methodof claim 1, wherein the particles have a mean pore volume in the rangeof about 0.1 to about 2.5 cm/g.
 5. The method of claim 1, wherein theparticles have a mean pore diameter in the range of about 100 to about1000 Angstroms.
 6. The method of claim 1, wherein the chromatographycolumn is kept in a temperature range of about 5° C. to about 85° C. 7.The method of claim 1, wherein the modifier added to the liquid CO₂ in aconstant or gradient mode or both over an elution period or a fractionthereof.
 8. The method of claim 7, wherein the modifier is a polarwater-miscible organic solvent selected from the group consisting ofmethanol, ethanol or isopropanol, acetonitrile, acetone,tetrahydrofuran, mixtures thereof, and mixtures of water and any ofthese solvents.
 9. The method of claim 7, wherein the gradient modecomprises increasing or decreasing flow volume of the modifier.
 10. Themethod of claim 7, wherein the gradient mode comprises increasing theflow volume of the modifier from about 0% to about 50% (v/v CO₂) or anyintervals therebetween.
 11. The method of claim 7, wherein the gradientmode comprises increasing the flow volume of the modified from about 0%to about 25% (v/v CO₂).
 12. The method of claim 1, wherein the elutionperiod is less than 5 minutes.
 13. The method of claim 1, wherein theliquid CO₂ is in a supercritical state or subcritical state or both. 14.The method of claim 1, wherein the detection comprises determining thelevels or the presence or absence of the one or more PAs.
 15. The methodof claim 1, wherein the detection is by way of a mass spectrometer;Evaporative Light Scattering (ELS) detector, Circular Dichroism (CD)detector, Flame Ionization Detector (FID) or a photodiode array detector(PDA).
 16. The method of claim 1, wherein the sample comprises apackaging material or a solution or an extract thereof.
 17. The methodof claim 1, wherein the chromatography column is part of achromatography system comprising a pre-column mobile phase dwell volumeof about 100 μL to about 500 μL; wherein said pre-column mobile phasedwell volume is the volume of the mobile phase present in a fluidicconnection between a junction at which the CO₂ and the modifiers aremixed and the head of the chromatography column.
 18. The method of claim1, wherein the one or more PAs are eluted from the chromatography columnby the mobile phase with a flow rate of about 1 to 4 mL/min.
 19. Themethod of claim 1, wherein the chromatography column has a length ofabout 50 to 150 mm and an internal diameter about 2 to 4 mm.
 20. Amethod for detecting one or more PAs comprising: (1) providing a samplecomprising one or more PAs for analysis; wherein the sample is preparedwith, extracted or dissolved in a diluent comprising at least 60%organic solvent, with the proviso that the sample is not subject to asolvent exchange step; (2) applying the sample to a chromatographysystem comprising: (a) a column with a solid stationary phase comprisingsilica particles having silanol groups on a surface of the silicaparticles and having a mean particle size of about 0.5 to about 3.5microns, wherein said column has a length of about 50 to 150 mm and aninternal diameter about 2 to 4 mm, and wherein the solid stationaryphase retains said one or more PAs; (b) a pre-column mobile phase dwellvolume of about 75 μL to about 500 μL; wherein said pre-column dwellvolume comprises a volume within a fluidic connection between a junctionat which two or more mobile phase solvents including CO₂ and a modifierare mixed to head of the column; and (c) a post-column mobile phasedwell volume of about 10 μL to about 450 μL; wherein said pose-columndwell volume comprises a volume within a tubular connection between theend of the column and a detector; (3) eluting the one or more PAs fromthe chromatography column by a mobile phase comprising a mixture of CO₂and a modifier to form one or more eluted PAs, wherein the mobile phasehas a flow rate of about 1 to 4 mL/min; and (4) detecting said one ormore eluted PAs.
 21. A kit for performing analysis or detecting one ormore PAs in a sample comprising: a sample preparation device forpreparing the sample comprising one or more PAs for analysis; whereinthe sample is prepared with, extracted or dissolved in a diluentcomprising at least about 60% organic solvent, with the proviso that thesample is analyzed without a solvent exchange step; a chromatographycolumn with a solid stationary phase comprising silica particles havingsilanol groups on a surface of the silica particles and having a meanparticle size of 0.5 to 3.5 microns; and one or more standards forcalibrating and facilitating the analysis and detection or the one ormore PAs.